Pathogenic yeast C. neoformans can disseminate through the blood stream and cause devastating meningitis. The mechanism transversal of C. neoformans across the blood-brain barrier (BBB) to cause the central nervous system (CNS) infection is largely unknown. The goal of this project is to continue investigating how C. neoformans enters into human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In the last grant period, we found that C. neoformans could induce morphological changes in HBMEC via cytoskeleton reorganization. We demonstrated that C. neoformans CPS1 encoded hyaluronic acid synthase. We also verified that CPS1 was required for C. neoformans binding to HBMEC using an in vitro BBB model. Furthermore, our studies showed that CD44 was the primary receptor on HBMEC for C. neoformans adhesion. Upon C. neoformans binding to the HBMEC, host CD44 translocated to the membrane rafts and surrounded the yeast entry site. Either CPS1 deletion in C. neoformans or CD44- knockout on HBMEC significantly impaired the yeast infection. We also observed that yeast binding and/or invasion was considerably reduced in the filipin-, GF109203X-, cytochalasin D- treated HBMEC. Filipin extracts cholesterol and caveolin on the membrane rafts, GF109203X is a Protein Kinase C (PKC) inhibitor, and cytochalasin D is a F-actin disrupting reagent. Dominant-negative PKCa also inhibited yeast invasion into HBMEC. The results suggest that the integrity of membrane rafts, functional PKCa, and F-actin were necessary for yeast invasion. Based on the above observations, we hypothesize that CD44-elicited signaling and induced cytoskeleton reorganization are required for yeast entry into HBMEC. We will explore the mechanisms of C. neoformans invasion by the following Aims: (1) To determine the CD44-elicited signaling during the C. neoformans invasion, (2) To examine how C. neoformans induces cytoskeleton reorganization on HBMEC and its relationship to yeast infection, and (3) To evaluate the role of CD44 during C. neoformans invasion in mouse models. In our previous grant period, we have demonstrated the adhesion step of pathogen-host interaction. In this grant period, we will further characterize the molecular events at the internalization step. These studies are related to the clinical observations that a significant number of patients suffer severe meningitis and eventually succumb to this pathogen. The information derived from the studies is expected to be helpful in the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.